Publications by authors named "Pascal Leblanc"

25 Publications

  • Page 1 of 1

Clinical and Molecular Landscape of ALS Patients with Mutations: Novel Pathogenic Variants and Novel Phenotypes. A Single ALS Center Study.

Int J Mol Sci 2020 Sep 16;21(18). Epub 2020 Sep 16.

Laboratoire de Biochimie et Biologie Moleculaire, CHU Nimes, Nimes, Motoneuron Disease: Pathophysiology and Therapy, INM, University Montpellier, 30029 Nîmes CEDEX 9, France.

Mutations in the copper zinc superoxide dismutase 1 () gene are the second most frequent cause of familial amyotrophic lateral sclerosis (ALS). Nearly 200 mutations of this gene have been described so far. We report all pathogenic variants identified in patients followed in the single ALS center of Lyon, France, between 2010 and 2020. Twelve patients from 11 unrelated families are described, including two families with the not yet described and mutations. Splice site mutations were detected in two families. We discuss implications concerning genetic screening of gene in familial and sporadic ALS.
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http://dx.doi.org/10.3390/ijms21186807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554847PMC
September 2020

Electrophysiological Characterization of C9ORF72-Associated Amyotrophic Lateral Sclerosis: A Retrospective Study.

Eur Neurol 2019 4;82(4-6):106-112. Epub 2020 Feb 4.

ALS Center, Hospices Civils de Lyon, Hôpital Neurologique Pierre Wertheimer, Service de Neurologie C et Service d'explorations Fonctionnelles Neurologiques, Bron, France.

Objective: C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS). The aim of the present study was to determine whether C9ORF72-associated ALS (C9-ALS) patients present distinctive electrophysiological characteristics that could differentiate them from non C9ORF72-associated ALS (nonC9-ALS) patients.

Methods: Clinical and electrodiagnostic data from C9-ALS patients and nonC9-ALS patients were collected retrospectively. For electroneuromyography, the mean values of motor conduction, myography, and the mean values of sensory conduction were considered. Furthermore, the proportion of ALS patients with electrophysiological sensory neuropathy was determined.

Results: No significant difference was observed between 31 C9-ALS patients and 22 nonC9-ALS patients for mean motor conduction and myography. For sensory conduction analyses, mean sensory conduction was not significantly different between both groups. In total, 38% of -C9-ALS patient and 21% of nonC9-ALS patients presented electrophysiological sensory neuropathy (p = 0.33). In -C9-ALS patients with electrophysiological sensory neuropathy, 80% (8/10) were male and 67% (6/9) presented spinal onset compare to 25% (4/16, p = 0.014) male and 25% (4/16, p = 0.087) with spinal onset in those without electrophysiological sensory neuropathy.

Conclusion: Although not different from nonC9-ALS, these results suggest that sensory involvement is a frequent feature of C9-ALS patients, expanding the phenotype of the disease beyond the motor and cognitive domains.
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http://dx.doi.org/10.1159/000505777DOI Listing
June 2020

Necrotizing Soft Tissue Infection Staphylococcus aureus but not S. pyogenes Isolates Display High Rates of Internalization and Cytotoxicity Toward Human Myoblasts.

J Infect Dis 2019 07;220(4):710-719

Centre International de Recherche en Infectiologie, Université de Lyon; Inserm U1111; Ecole Normale Supérieure de Lyon; Université Lyon 1, CNRS, UMR5308; Lyon, France.

Background: Necrotizing soft tissue infections (NSTIs) caused by group A Streptococcus (GAS) and occasionally by Staphylococcus aureus (SA) frequently involve the deep fascia and often lead to muscle necrosis.

Methods: To assess the pathogenicity of GAS and S. aureus for muscles in comparison to keratinocytes, adhesion and invasion of NSTI-GAS and NSTI-SA isolates were assessed in these cells. Bloodstream infections (BSI-SA) and noninvasive coagulase-negative staphylococci (CNS) isolates were used as controls.

Results: NSTI-SA and BSI-SA exhibited stronger internalization into human keratinocytes and myoblasts than NSTI-GAS or CNS. S. aureus internalization reached over 30% in human myoblasts due to a higher percentage of infected myoblasts (>11%) as compared to keratinocytes (<3%). Higher cytotoxicity for myoblasts of NSTI-SA as compared to BSI-SA was attributed to higher levels of psmα and RNAIII transcripts in NSTI-SA. However, the 2 groups were not discriminated at the genomic level. The cellular basis of high internalization rate in myoblasts was attributed to higher expression of α5β1 integrin in myoblasts. Major contribution of FnbpAB-integrin α5β1 pathway to internalization was confirmed by isogenic mutants.

Conclusions: Our findings suggest a factor in NSTI-SA severity is the strong invasiveness of S. aureus in muscle cells, a property not shared by NSTI-GAS isolates.
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http://dx.doi.org/10.1093/infdis/jiz167DOI Listing
July 2019

Phosphorylated and aggregated TDP-43 with seeding properties are induced upon mutant Huntingtin (mHtt) polyglutamine expression in human cellular models.

Cell Mol Life Sci 2019 Jul 12;76(13):2615-2632. Epub 2019 Mar 12.

Institut NeuroMyoGène, CNRS UMR5310, INSERM U1217, Faculté de Médecine Rockefeller, Université Claude Bernard Lyon I, 8 Avenue Rockefeller, 69373, Lyon Cedex 08, France.

The Tar DNA-Binding Protein 43 (TDP-43) and its phosphorylated isoform (pTDP-43) are the major components associated with ubiquitin positive/Tau-negative inclusions found in neurons and glial cells of patients suffering of amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration-TDP-43 (FTLD-TDP). Many studies have revealed that TDP-43 is also in the protein inclusions associated with neurodegenerative conditions other than ALS and FTLD-TDP, thus suggesting that this protein may be involved in the pathogenesis of a variety of neurological disorders. In brains of Huntington-affected patients, pTDP-43 aggregates were shown to co-localize with mutant Huntingtin (mHtt) inclusions. Here, we show that expression of mHtt carrying 80-97 polyglutamines repeats in human cell cultures induces the aggregation and the phosphorylation of endogenous TDP-43, whereas non-pathological Htt with 25 polyglutamines repeats has no effect. Mutant Htt aggregation precedes accumulation of pTDP-43 and pTDP-43 co-localizes with mHtt inclusions reminding what it was previously described in brains of Huntington-affected patients. Detergent-insoluble fractions from cells expressing mHtt and containing mHtt-pTDP-43 co-aggregates can function as seeds for further TDP-43 aggregation in human cell culture. The human cellular prion protein PrP was previously identified as a negative modulator of mHtt aggregation; here, we show that PrP-mediated reduction of mHtt aggregation is tightly correlated with a decrease of TDP-43 aggregation and phosphorylation, thus confirming the close relationships between TDP-43 and mHtt.
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http://dx.doi.org/10.1007/s00018-019-03059-8DOI Listing
July 2019

Development of an automated capillary nano-immunoassay-Simple Western assay-to quantify total TDP43 protein in human platelet samples.

Anal Bioanal Chem 2019 Jan 29;411(1):267-275. Epub 2018 Oct 29.

Neurochemistry Laboratory, Biochemistry Department, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, 59 Bd Pinel, 69677, Bron, France.

Frontotemporal lobar degeneration syndrome is the second cause of young-onset dementia. Unfortunately, reliable biomarkers are currently lacking for the diagnosis of this disease. As TDP43 protein is one of the proteins pathologically involved in frontotemporal lobar degeneration, many studies have been performed to assess TDP43 protein diagnostic performances. Mixed results were obtained using cerebrospinal fluid and plasma samples so far. The aim of the study was to develop an automated capillary nano-immunoassay-Simple Western assay-to detect and quantify TDP43 protein simultaneously in human blood-based samples. Simple Western assay was developed with two different cell lysates used as positive controls and was compared to Western blot. TDP43 protein profiles in plasma samples were disappointing, as they were discordant to our positive controls. On the contrary, similar TDP43 patterns were obtained between platelet samples and cell lysates using both assays. Simple Western assay provided good quantitative performances in platelet samples: a linearity of signals could be observed (r = 0.994), associated to a within-run variability at 5.7%. Preliminary results based on a cohort of patients suffering from frontotemporal lobar degeneration showed large inter-individual variations superior to Simple Western's analytical variability. Simple Western assay seems to be suitable for detecting and quantifying TDP43 protein in platelet samples, providing a potential candidate biomarker in this disease. Further confirmation studies should now be performed on larger cohorts of patients to assess diagnostic performances of TDP43 protein in platelet samples.
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http://dx.doi.org/10.1007/s00216-018-1437-4DOI Listing
January 2019

Isotopic Evidence for Disrupted Copper Metabolism in Amyotrophic Lateral Sclerosis.

iScience 2018 Aug 1;6:264-271. Epub 2018 Aug 1.

Université de Lyon, ENS de Lyon, CNRS, LGL-TPE, 69007 Lyon, France. Electronic address:

Redox-active metals are thought to be implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). To address this point, we measured the concentrations of 12 elements and, for the first time, the stable isotope compositions of copper (redox-active) and zinc (redox-inactive) in human cerebrospinal fluids of 31 patients with ALS, 11 age-matched controls (CTRL), and 14 patients with Alzheimer disease. We first show that metal concentrations weakly discriminate patients with ALS from the two other groups. We then report that zinc isotopic compositions are similar in the three groups, but that patients with ALS have significantly 65copper-enriched isotopic compositions relative to CTRL and patients with AD. This result unambiguously demonstrates that copper is implicated in ALS. We suggest that this copper isotopic signature may result from abnormal protein aggregation in the brain parenchyma, and propose that isotopic analysis is a potential tool that may help unraveling the molecular mechanisms at work in ALS.
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http://dx.doi.org/10.1016/j.isci.2018.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137708PMC
August 2018

Correction to: Cellular mechanisms responsible for cell-to-cell spreading of prions.

Cell Mol Life Sci 2018 07;75(14):2575

Insitut NeuroMyoGène, CNRS UMR5310, INSERM U1217, Faculté de Médecine Rockefeller, Université Claude Bernard Lyon I, 8 Avenue Rockefeller, 69373, Lyon Cedex 08, France.

In the original publication, part of acknowledgement text was missing. The complete acknowledgement section should read as follows.
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http://dx.doi.org/10.1007/s00018-018-2853-5DOI Listing
July 2018

Cellular mechanisms responsible for cell-to-cell spreading of prions.

Cell Mol Life Sci 2018 Jul 14;75(14):2557-2574. Epub 2018 May 14.

Insitut NeuroMyoGène, CNRS UMR5310, INSERM U1217, Faculté de Médecine Rockefeller, Université Claude Bernard Lyon I, 8 Avenue Rockefeller, 69373, Lyon Cedex 08, France.

Prions are infectious agents that cause fatal neurodegenerative diseases. Current evidence indicates that they are essentially composed of an abnormally folded protein (PrP). These abnormal aggregated PrP species multiply in infected cells by recruiting and converting the host PrP protein into new PrP. How prions move from cell to cell and progressively spread across the infected tissue is of crucial importance and may provide experimental opportunity to delay the progression of the disease. In infected cells, different mechanisms have been identified, including release of infectious extracellular vesicles and intercellular transfer of PrP-containing organelles through tunneling nanotubes. These findings should allow manipulation of the intracellular trafficking events targeting PrP in these particular subcellular compartments to experimentally address the relative contribution of these mechanisms to in vivo prion pathogenesis. In addition, such information may prompt further experimental strategies to decipher the causal roles of protein misfolding and aggregation in other human neurodegenerative diseases.
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http://dx.doi.org/10.1007/s00018-018-2823-yDOI Listing
July 2018

Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages.

Stem Cell Reports 2017 12 30;9(6):2018-2033. Epub 2017 Nov 30.

Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, PRES Sorbonne-Paris-Cité, 75014 Paris, France; Institut NeuroMyoGène, Université Lyon, Université Claude Bernard Lyon 1, INSERM U1217, CNRS UMR5534, 16 rue Raphael Dubois, Bâtiment Gregor Mendel, 69100 Villeurbanne, France. Electronic address:

In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785732PMC
December 2017

Expression of Heterologous PrP and Prion Propagation in RK13 Cells.

Methods Mol Biol 2017 ;1658:95-104

INRA, UMR 1225, IHAP, 31076, Toulouse, France.

Cultured cells are valuable models to study prion infections at the cellular level. Unfortunately, the vast majority of cell lines are resistant to the propagation of prion agents. The rabbit epithelial RK13 cell line is among the few cell lines permissive to prion infection. When genetically engineered to express heterologous PrP proteins, RK13 cells become permissive to several strains of prions from various animal species. Here, we describe the generation of stable RK13 cell clones expressing a heterologous PrP protein in an inducible manner, the establishment and maintenance of chronically infected cultures, and the selection of cell clones suitable for cell-based titration of prions.
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http://dx.doi.org/10.1007/978-1-4939-7244-9_8DOI Listing
May 2018

[Endogenous retroviral sequences in the human genome can play a physiological or pathological role].

Med Sci (Paris) 2017 Apr 12;33(4):397-403. Epub 2017 May 12.

GeNeuro Innovation, Bioparc Laënnec, 60, avenue Rockefeller, 69008 Lyon, France - Université Lyon-1, Faculté de Médecine Laënnec, 69008 Lyon, France - GeNeuro, 18, chemin des Aulx, 1228 Plan-Les-Ouates, Genève, Suisse.

Human endogenous retroviruses (HERV) represent a large part of our genome and the few elements that have retained a potential of expression still remain "dormant" in physiological conditions. In some instances, they can be awakened by environmental factors activating their expression. The best studied conditions of HERV activation are infections caused by microorganisms such as viruses of the Herpesvirus family. This activation can thus lead to the expression of pathogenic proteins such as envelope proteins belonging to the HERV-W and HERV-K families, respectively involved in Multiple Sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Endogenous retroviral proteins can also acquire a physiological function beneficial for humans. This is the case of Syncytin-1 from the HERV-W family, that is involved in placenta formation.
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http://dx.doi.org/10.1051/medsci/20173304009DOI Listing
April 2017

Isolation of Exosomes and Microvesicles from Cell Culture Systems to Study Prion Transmission.

Methods Mol Biol 2017 ;1545:153-176

IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France.

Extracellular vesicles (EVs) are composed of microvesicles and exosomes. Exosomes are small membrane vesicles (40-120 nm sized) of endosomal origin released in the extracellular medium from cells when multivesicular bodies fuse with the plasma membrane, whereas microvesicles (i.e., shedding vesicles, 100 nm to 1 μm sized) bud from the plasma membrane. Exosomes and microvesicles carry functional proteins and nucleic acids (especially mRNAs and microRNAs) that can be transferred to surrounding cells and tissues and can impact multiple dimensions of the cellular life. Most of the cells, if not all, from neuronal to immune cells, release exosomes and microvesicles in the extracellular medium, and all biological fluids including blood (serum/plasma), urine, cerebrospinal fluid, and saliva contain EVs.Prion-infected cultured cells are known to secrete infectivity into their environment. We characterized this cell-free form of prions and showed that infectivity was associated with exosomes. Since exosomes are produced by a variety of cells, including cells that actively accumulate prions, they could be a vehicle for infectivity in body fluids and could participate to the dissemination of prions in the organism. In addition, such infectious exosomes also represent a natural, simple, biological material to get key information on the abnormal PrP forms associated with infectivity.In this chapter, we describe first a method that allows exosomes and microvesicles isolation from prion-infected cell cultures and in a second time the strategies to characterize the prions containing exosomes and their ability to disseminate the prion agent.
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http://dx.doi.org/10.1007/978-1-4939-6728-5_11DOI Listing
February 2018

Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway.

Cell Mol Life Sci 2015 Nov 6;72(22):4409-27. Epub 2015 Jun 6.

CNRS, UMR5239, Laboratoire de Biologie Moléculaire de la Cellule (LBMC), ENS Lyon, 46 allée d'Italie, 69364, Lyon 7, France.

Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation.
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http://dx.doi.org/10.1007/s00018-015-1945-8DOI Listing
November 2015

Prion strains are differentially released through the exosomal pathway.

Cell Mol Life Sci 2015 Mar 18;72(6):1185-96. Epub 2014 Sep 18.

INRA, UMR 1225, IHAP, 31076, Toulouse, France.

Cell-to-cell transfer of prions is a crucial step in the spreading of prion infection through infected tissue. At the cellular level, several distinct pathways including direct cell-cell contacts and release of various types of infectious extracellular vesicles have been described that may potentially lead to infection of naïve cells. The relative contribution of these pathways and whether they may vary depending on the prion strain and/or on the infected cell type are not yet known. In this study we used a single cell type (RK13) infected with three different prion strains. We showed that in each case, most of the extracellular prions resulted from active cell secretion through the exosomal pathway. Further, quantitative analysis of secreted infectivity indicated that the proportion of prions eventually secreted was dramatically dependent on the prion strain. Our data also highlight that infectious exosomes secreted from cultured cells might represent a biologically pertinent material for spiking experiments. Also discussed is the appealing possibility that abnormal PrP from different prion strains may differentially interact with the cellular machinery to promote secretion.
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http://dx.doi.org/10.1007/s00018-014-1735-8DOI Listing
March 2015

Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

Nucleic Acids Res 2014 Sep 4;42(15):9700-16. Epub 2014 Aug 4.

CIRI, International Center for Infectiology Research, Oncogenic Herpesviruses team, Université de Lyon, Lyon 69364, France Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon 69364, France CIRI, International Center for Infectiology Research, Cell Biology of Viral Infections team, Université de Lyon, Lyon 69364, France INSERM, U1111, Lyon 69364, France CNRS, UMR5308, Lyon 69364, France

The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.
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http://dx.doi.org/10.1093/nar/gku697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150796PMC
September 2014

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells.

Proc Natl Acad Sci U S A 2013 Jun 10;110(26):10658-63. Epub 2013 Jun 10.

Institut Curie, and Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique, F-75248 Paris, France.

Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aβ peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.
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http://dx.doi.org/10.1073/pnas.1220748110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696817PMC
June 2013

Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes.

Nat Struct Mol Biol 2012 Apr 8;19(5):517-24, S1. Epub 2012 Apr 8.

Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Strasbourg, France.

Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrP(C) (or PRNP) has poorly understood physiological functions. Whereas most PrP(C) is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrP(C) that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrP(C) variant orients the N-terminal 'octarepeat' domain into the cytoplasm. Here we demonstrate that the octarepeat domain of human PrP(C) contains GW/WG motifs that bind Argonaute (AGO) proteins, the essential components of microRNA (miRNA)-induced silencing complexes (miRISCs). Transmembrane PrP(C) preferentially binds AGO, and PrP(C) promotes formation or stability of miRISC effector complexes containing the trinucleotide repeat-containing gene 6 proteins (TNRC6) and miRNA-repressed mRNA. Accordingly, effective repression of several miRNA targets requires PrP(C). We propose that dynamic interactions between PrP(C)-enriched endosomes and subcellular foci of AGO underpin these effects.
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http://dx.doi.org/10.1038/nsmb.2273DOI Listing
April 2012

Co-infection with the friend retrovirus and mouse scrapie does not alter prion disease pathogenesis in susceptible mice.

PLoS One 2012 25;7(1):e30872. Epub 2012 Jan 25.

Université de Lyon, Lyon, France.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030872PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266293PMC
June 2012

Functional mechanisms of the cellular prion protein (PrP(C)) associated anti-HIV-1 properties.

Cell Mol Life Sci 2012 Apr 11;69(8):1331-52. Epub 2011 Nov 11.

Université de Lyon, France.

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.
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http://dx.doi.org/10.1007/s00018-011-0879-zDOI Listing
April 2012

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells.

Prion 2010 Oct-Dec;4(4):292-301. Epub 2010 Oct 27.

CNRS, Institut de Génétique Humaine UPR1142, Université Montpellier and Institut de Recherches en Biothérapie (IRB), Biochimie-Protéomique Clinique, CHU de Montpellier, Montpellier, France.

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP(C)) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP(C) is converted into an abnormally folded isoform, called PrP(Sc), that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP(C) and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrP(Sc) production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.
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http://dx.doi.org/10.4161/pri.4.4.13435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268962PMC
June 2011

Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles.

Biol Cell 2008 Oct;100(10):603-15

LaboRétro INSERM U758, Unité de Virologie Humaine, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France.

Background Information: TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrP(Sc), a conformationally altered isoform of the normal prion protein (PrP(C)), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a).

Results: The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome-like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome-like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs.

Conclusions: The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.
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http://dx.doi.org/10.1042/BC20080025DOI Listing
October 2008

Retrovirus infection strongly enhances scrapie infectivity release in cell culture.

EMBO J 2006 Jun 25;25(12):2674-85. Epub 2006 May 25.

LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France.

Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.
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http://dx.doi.org/10.1038/sj.emboj.7601162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1500854PMC
June 2006

Analysis of the interactions between HIV-1 and the cellular prion protein in a human cell line.

J Mol Biol 2004 Apr;337(4):1035-51

INSERM U412 Ecole Normale Supérieure de LYON, Lyon, France.

The cellular prion protein (PrP(c)) is highly conserved in mammals and expressed widely in different tissues but its physiological role remains elusive. Recently, the human PrP(c) was shown to possess nucleic acid binding and chaperoning properties similar to human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, a key viral factor in virus structure and replication. These findings prompted us to determine if PrP(c) could influence HIV-1 replication. We used the human 293T cell line as a model system, since only a very low level of PrP(c) accumulates in these cells. Expression of PrP at a high level resulted in a specific decrease of HIV-1 Env and Vpr expression. Despite similar levels of intracellular Gag, virus production was reduced by eightfold and infectivity by three- to fourfold in the presence of PrP(c). A PrP(c) mutant lacking the glycosylphosphatidylinositol (GPI) anchor peptide did not impair HIV-1 production, suggesting that PrP(c) trafficking is critical for this inhibitory effect. Coexpressing HIV-1 and PrP(c) in these cells also caused a fraction of PrP(c) to become partially proteinase K-resistant (PrP(res)), further illustrating the interactions between HIV-1 and PrP(c).
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http://dx.doi.org/10.1016/j.jmb.2004.02.007DOI Listing
April 2004

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

C R Biol 2002 Jan;325(1):17-23

Laboretro, unité de virologie humaine, Inserm-ENS U412, ENS de Lyon, 46, allée d'Italie, 69364 Lyon, France.

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.
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http://dx.doi.org/10.1016/s1631-0691(02)01388-4DOI Listing
January 2002